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BASIC RESEARCH
Year : 2015  |  Volume : 60  |  Issue : 6  |  Page : 544-548
Vitamin D receptor expression in vitiligo


1 Department of Dermatology, Faculty of Medicine, Beni Suef University, Beni Suef, Egypt
2 Department of Biochemistry, Faculty of Medicine, Cairo University, Cairo, Egypt

Date of Web Publication5-Nov-2015

Correspondence Address:
Reham William Doss
Department of Dermatology, Faculty of Medicine, Beni-Suef University, Mohammed Hassan Street, Beni-Suef
Egypt
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0019-5154.169123

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   Abstract 

Background: Vitiligo is a progressive depigmenting disorder characterized by loss of functional melanocytes from the epidermis. The etiopathogenesis of vitiligo is still unclear. Vitamin D has stimulatory effects on melanocytes and acts through its nuclear Vitamin D receptor (VDR) on target cells. Aims and Objectives: The purpose of this study was to declare the role of Vitamin D in the pathogenesis of vitiligo. Materials and Methods: This case-control study included 30 vitiligo patients and 30 age, gender-matched healthy controls. Blood samples were withdrawn from the study subjects, and the serum 25(OH) D level was determined by an enzyme-linked immunosorbent assay technique. Serum 25(OH) D levels were divided into: Normal or sufficient (΃30 ng/ml), insufficient (< 30-> 20 ng/ml), and deficient (ͳ20 ng/ml) levels. Skin biopsies were obtained from the depigmented lesions and clinically normal skin of vitiligo patients and from the controls, and VDR gene expression was determined using real-time polymerase chain reaction. Results: Only 10 patients with vitiligo (33.3%) had sufficient serum 25(OH) D levels (΃30 ng/ml), 12 patients (40%) had insufficient levels, and 8 patients (26.7%) had deficient levels. On the other hand, most of the controls (96.7%) had sufficient levels. The mean serum 25(OH) D level in patients was significantly decreased compared to controls (P < 0.001). The VDR-mRNA expression was also significantly decreased in lesional and nonlesional skin of patients compared to controls (P < 0.001, P < 0.001, respectively). Conclusion: Vitamin D deficiency influences the extent of vitiligo and could contribute to the pathogenesis of vitiligo through its immunomodulatory role and its role in melanogenesis.


Keywords: 25-hydroxy Vitamin D, enzyme-linked immunosorbent assay, real-time polymerase chain reaction, Vitamin D receptor, Vitiligo


How to cite this article:
Doss RW, El-Rifaie AA, Gohary YM, Rashed LA. Vitamin D receptor expression in vitiligo. Indian J Dermatol 2015;60:544-8

How to cite this URL:
Doss RW, El-Rifaie AA, Gohary YM, Rashed LA. Vitamin D receptor expression in vitiligo. Indian J Dermatol [serial online] 2015 [cited 2021 Apr 15];60:544-8. Available from: https://www.e-ijd.org/text.asp?2015/60/6/544/169123

What was known?

  • Vitiligo is characterized by loss of functioning melanocytes
  • Vitamin D has stimulatory effects on melanocytes.



   Introduction Top


Vitiligo is a common pigmentary disorder characterized by well-demarcated depigmented patches or macules of different shapes and sizes and caused by the destruction of functional melanocytes in the involved epidermis. [1]

Vitamin D is a steroid hormone that plays an important role in calcium homeostasis. The main source is an endogenous synthesis in the skin, which is induced by ultraviolet B (UVB) radiation. [2]

Vitamin D exerts its effect through a nuclear hormone receptor called the Vitamin D receptor (VDR). [3] Melanocytes express the VDRs, which take part in the regulation of melanin synthesis. [4] Several researchers pointed at the role of Vitamin D in melanogenesis. For instance, Tomita et al., [5] found that Vitamin D 3 increased the tyrosinase content of cultured human melanocytes and Watabe et al. [6] observed that 1,25(OH) 2 D 3 might stimulate the differentiation of immature melanocyte precursors.

The aim of this study was to evaluate Vitamin D role in the pathogenesis of vitiligo through the evaluation of Vitamin D serum level and VDR expression in tissue biopsies from the normal and vitiliginous skin.


   Materials and Methods Top


This case-control study included 30 patients with vitiligo and 30 age and sex matched healthy controls. The patients and controls were of Fitzpatrick skin type IV recruited from individuals attending the outpatient clinic of the Beni-Suef University Hospitals in the period (from June 1, 2013 to September 1, 2013).

Exclusion criteria included the use of any drugs that could alter the outcome of the study such as topical or systemic Vitamin D or calcium, systemic steroids, weight loss drugs, cholesterol lowering drugs, thiazide diuretics, and sunscreens in the last 6 months.

Pregnant, lactating females, obese, smokers, subjects with malabsorption disorders, other autoimmune diseases, kidney diseases, and subjects receiving phototherapy the last 6 months were also excluded from the study.

Patient information was collected by one dermatologist including age, sex, type of vitiligo (generalized, localized or universal), [7] affected body surface area (BSA) according to the rule of nines, [8] and disease activity determined by the vitiligo disease activity (VIDA) score. [9]

The purpose of the study was explained to each patient, and written consent was taken. The study protocol conforms to the ethical guidelines of the 1975 Declaration of Helsinki as reflected in a priori approval by the Institution's Human Research Committee.

Estimation of Vitamin D in serum using enzyme-linked immunosorbent assay

Blood samples were withdrawn and left to clot for 20 min, then centrifuged at 12,000 rpm for 10 min, then the separated serum was kept frozen till analysis. Serum samples were examined for 25(OH) D levels by enzyme-linked immunosorbent assay (ELISA) using the kit supplied by Immunodiagnostic USA. The samples were incubated with the detection antibody after the extraction step. Then peroxidase-conjugated antibody was added into the microplate well forming a complex of 25(OH) D - detection antibody - peroxidase conjugate. Tetramethylbenzidine was used as a substrate. The color density developed is proportional to Vitamin D concentration. Finally, to terminate the reaction, stop solution was added, and the microplate was read by ELISA reader at 450 nm. [10]

Serum levels of 25(OH) D were divided into sufficient (≥30 ng/ml); insufficient (20-30 ng/ml) and deficient (≤20 ng/ml) levels. [11]

Detection of Vitamin D receptor expression in tissue

RNA extraction


Total RNA was isolated from skin tissue homogenates using RNeasy Purification Reagent (Qiagen, Valencia, CA, USA) according to manufacturer's instruction. The purity (A260/A280 ratio) and the concentration of RNA were obtained using spectrophotometry (GeneQuant 1300, Uppsala, Sweden). RNA quality was confirmed by gel electrophoresis.

cDNA synthesis

First-strand cDNA was synthesized from 4 μg of total RNA using an Oligo (dT) 12-18 primer and Superscript II RNase reverse transcriptase, this mixture was incubated at 42°C for 1 h, the kit was supplied by SuperScript choice system (Life Technologies, Breda, The Netherlands).

Real-time quantitative polymerase chain reaction

Real-time polymerase chain reaction (RT-PCR) amplification was carried out using 10 μL amplification mixtures containing Power SYBR Green PCR master mix (Applied Biosystems, Foster City, CA, USA), equivalent to 8 ng of reverse-transcribed RNA and 300 nM primers; the sequences of PCR primer pairs used for each gene are shown in [Table 1]. Reactions were run on an ABI PRISM 7900 HT detection system (Applied Biosystems); PCR reactions consisting of 95°C for 10 min (1 cycle), 94°C for 15 s, and 60°C for 1 min (40 cycles). Data were analyzed with the ABI Prism sequence detection system software and quantified using the v1·7 Sequence detection software from PE Biosystems (Foster City, CA, USA). Relative expression of studied genes was calculated using the comparative threshold cycle method. All values were normalized to the GAPDH genes. [12]
Table 1: Primer sequences used for quantitative RT-PCR


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Statistical analysis

Data were analyzed using the software, Statistical Package for Social Science, version 18. A frequency distribution with its percentage and descriptive statistics with mean and standard deviation (SD) were calculated. Chi-square, t-test, correlations were carried out whenever needed. P < 0.05 was considered significant.


   Results Top


The gender ratio, age were not substantially different for each variable among patients with vitiligo (10 women, 20 men; mean ± SD age 32.5 ± 14.6 years), and healthy controls (14 women, 16 men; mean ± SD age 28 ± 5.7) (P = 0.4, P = 0.2, respectively). Clinical data of participants are presented in [Table 2].
Table 2: Demographic data, clinical characteristics of vitiligo patients, and controls


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Serum 25(OH) D Levels

The mean 25-hydroxy Vitamin D (25(OH) D) level in controls was almost twice that of patients (P < 0.001) [Figure 1].
Figure 1: Reduced mean level of 25(OH)D in vitiligo patients when compared to control subjects (P < 0.001)

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We found that 10 patients with vitiligo (33.3%) versus almost all the controls (96.7%) had sufficient serum 25(OH) D levels; twelve patients (40%) versus only one control subject (3.3%) had insufficient levels; while eight patients (26.7%) and none of the controls had deficient 25(OH) D levels, which means that 20 patients (66.7%) versus only one control subject (3.3%) had 25(OH) levels below 30 ng/ml [Table 3] and [Figure 2].
Figure 2: Among 25(OH)D levels were divided into: normal or sufficient (≥30 ng/ml), insufficient (< 30->20 ng/ml), and deficient (≤20 ng/ml) levels. Only 10 patient with vitiligo (33.3%) had sufficient serum 25(OH)D levels (≥30 ng/ml) while 20 patients (66.7%) had levels below 30 ng/ml

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Table 3: Serum 25(OH)D levels and VDR expression in vitiligo patients, and controls


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Although the levels of 25(OH) were low among patients, we could find no relation with the age, sex, disease duration, family history of vitiligo, stress at onset, vitiligo type, or VIDA score with P > 0.05 in all.

When we made a cut-off at serum 25(OH) D level of 30 ng/ml; we found that patients with 25(OH) D levels ≥30 ng/ml had higher affected BSA compared with those with levels < 30 ng/ml (mean ± SD; 47.5 ± 33.7 versus 33.3 ± 31.5, respectively).

The tissue Vitamin D receptor expression

The mean VDR expression in the tissue biopsies of controls was more than 8 times higher than the lesional tissue biopsies of patients (P < 0.001) and 3 times higher than the nonlesional biopsies from patients (P < 0.001).

Surprisingly, the mean VDR expression in the nonlesional biopsies was also more than double the mean VDR expression in the lesional biopsies (P < 0.001) [Table 3].

Again, we found no relation between patient age, sex, disease duration, family history, stress at onset, vitiligo type, VIDA score or affected BSA with the VDR expression (both in lesional and nonlesional tissue biopsies) and P > 0.05 in all.

No significant difference existed in VDR expression (in lesional or nonlesional tissue biopsies) between patient group with 25(OH) D levels above 30 ng/ml and the patient group below 30 ng/ml and the P > 0.05.


   Discussion Top


The exact pathogenesis of vitiligo is unknown, but researchers suggested that an interplay of multiple factors as genetic, neural, oxidant-antioxidant, biochemical, minerals, and autoimmune process might induce vitiligo. [13]

Vitamin D is synthesized in the epidermal keratinocytes under the influence of UVB light. [14] Vitamin D compounds are known to influence melanogenesis [4],[5],[6] Moreover, topical Vitamin D analogs were used as monotherapy or in combination with phototherapy for the treatment of vitiligo. [15]

All the above mentioned prompted us to investigate the Vitamin D status in Egyptian vitiligo patients, and in order to do so we estimated the 25(OH) D levels in serum, which is a widely accepted indicator of overall Vitamin D status. [16] Serum 25(OH) D levels were divided into: Normal or sufficient (΃30 ng/ml), insufficient (<30->20 ng/ml), and deficient (≤20 ng/ml) levels.

Our results showed that the mean serum levels of 25(OH) D were markedly lower among cases than controls. We found that 26.7% of our patients had deficient 25(OH) levels. Moreover, 40% of patients had insufficient levels, which means that 66.7% of patients had 25(OH) D levels below 30 ng/ml.

Inconsistent with our results, Silverberg et al. [17] found that more than 68.9% of patients had serum levels of 25(OH) D below 30 ng/ml and, Saleh et al. [18] found that 97.5% of patients had deficient and also recommended levels of 25(OH) D.

On the contrary, Xu et al. [19] conducted a study over 171 Chinese patients and could detect no significant difference in the 25(OH) D levels between patients and controls.

The affected BSA was higher in patients with 25(OH) D level above 30 ng/ml compared to those with levels below 30 ng/ml, which means that the level of Vitamin D could influence the extent of the disease.

We propose that the low serum levels of Vitamin D in vitiligo patients could be a consequence of the disease, as well as a contributing factor, to the development of the disease.

As a consequence of the disease, patients suffering from vitiligo might have more psychological stress and tend not to expose their skin to the public, which would influence the Vitamin D synthesis.

On the other hand, deficient Vitamin D levels could contribute to the development of vitiligo through its immunomodulatory role, as well as its impact on melanogenesis.

A connection between some autoimmune diseases including type I diabetes mellitus, systemic lupus, psoriasis, and Vitamin D deficiency had been reported. [20] Besides, low levels of Vitamin D were also found in pemphigus vulgaris and bullous pemphigoid. [21]

The mechanism by which Vitamin D affects autoimmunity is not yet clear. It was found that 1,25(OH) 2 D 3 could inhibit the differentiation and maturation of dendritic cells; reduce the expression of major histocompatibility complex class II, and inhibit the secretion of proinflammatory cytokines including interleukin 1 (IL-1), IL-2, IL-6, interferon gamma, tumor necrosis factor alpha, IL-12, and also inhibit IL-6 and IL-17 secretion thus impeding Th17 function. [19] Some of these proinflammatory and proapoptotic cytokines were found to play a role in the pathogenesis of vitiligo. [22],[23]

As a consequence, we could suggest that low levels of Vitamin D might impair Th1 and Th17 pathways contributing to the development of vitiligo.

As mentioned before, Vitamin D compounds influence maturation, differentiation, proliferation, migration, and melanin production of melanocytes; the melanocyte response to melanogenic stimuli is modulated by calcium-dependent regulatory enzymes or transcription factors, which might indicate that Vitamin D is likely to have an additional role in melanogenesis through its calcemic effect.

The VDR gene polymorphism was suggested to influence the Vitamin D level and consequently play an important role in the genetic susceptibility to vitiligo. [24] This primed us to investigate the VDR expression in vitiligo patients.

To the best of our knowledge, this is the first study to estimate the expression of VDR in vitiligo patients. Our results showed that the expression of VDR in tissue biopsies from the controls was significantly higher than both the lesional and nonlesional tissue biopsies from patients.

The low expression of VDR in vitiligo patients compared to controls could be explained by the markedly low Vitamin D levels that could influence the VDR expression on keratinocytes. On the other hand, the absent melanocytes, which harbor the VDR from vitiligo lesions, could simply explain the markedly higher VDR expression in controls compared to lesional tissue biopsies.

Surprisingly, we found no difference in VDR expression in patients with 25(OH) D levels above 30 ng/ml and below 30 ng/ml, which might point at the role of other factors like genetic suitability in influencing the VDR expression in vitiligo. However, these results have to be confirmed on a larger study group.


   Recommendations Top


The screening of Vitamin D level for possible Vitamin D supplementation in patients of vitiligo before the starting of therapy is recommended and further studies that access the response to phototherapy in patients receiving Vitamin D supplementation are also recommended.

 
   References Top

1.
AlGhamdi KM, Kumar A. Depigmentation therapies for normal skin in vitiligo universalis. J Eur Acad Dermatol Venereol 2011;25:749-57.  Back to cited text no. 1
    
2.
Holick MF. Sunlight and vitamin D for bone health and prevention of autoimmune diseases, cancers, and cardiovascular disease. Am J Clin Nutr 2004;80:1678S-88.  Back to cited text no. 2
    
3.
Wang TT, Tavera-Mendoza LE, Laperriere D, Libby E, MacLeod NB, Nagai Y, et al. Large-scale in silico and microarray-based identification of direct 1,25-dihydroxyvitamin D3 target genes. Mol Endocrinol 2005;19:2685-95.  Back to cited text no. 3
    
4.
Abdel-Malek ZA, Ross R, Trinkle L, Swope V, Pike JW, Nordlund JJ. Hormonal effects of vitamin D3 on epidermal melanocytes. J Cell Physiol 1988;136:273-80.  Back to cited text no. 4
    
5.
Tomita Y, Torinuki W, Tagami H. Stimulation of human melanocytes by vitamin D3 possibly mediates skin pigmentation after sun exposure. J Invest Dermatol 1988;90:882-4.  Back to cited text no. 5
    
6.
Watabe H, Soma Y, Kawa Y, Ito M, Ooka S, Ohsumi K, et al. Differentiation of murine melanocyte precursors induced by 1,25-dihydroxyvitamin D3 is associated with the stimulation of endothelin B receptor expression. J Invest Dermatol 2002;119:583-9.  Back to cited text no. 6
    
7.
Ortonne J. Vitiligo and other disorders of hypopigmentation. In: Bolognia JL, Jorizzo JL, Rapini RP, editors. Dermatology. 2 nd ed. Spain: Elsevier; 2008. p. 913-20.  Back to cited text no. 7
    
8.
Kanthraj GR, Srinivas CR, Shenoi SD, Deshmukh RP, Suresh B. Comparison of computer-aided design and rule of nines methods in the evaluation of the extent of body involvement in cutaneous lesions. Arch Dermatol 1997;133:922-3.  Back to cited text no. 8
    
9.
Njoo MD, Das PK, Bos JD, Westerhof W. Association of the Köbner phenomenon with disease activity and therapeutic responsiveness in vitiligo vulgaris. Arch Dermatol 1999;135:407-13.  Back to cited text no. 9
    
10.
Wielders JP, Wijnberg FA. Preanalytical stability of 25(OH)-vitamin D3 in human blood or serum at room temperature: Solid as a rock. Clin Chem 2009;55:1584-5.  Back to cited text no. 10
    
11.
Bischoff-Ferrari HA. The 25-hydroxyvitamin D threshold for better health. J Steroid Biochem Mol Biol 2007;103:614-9.  Back to cited text no. 11
    
12.
Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C (T)) method. Methods 2001;25:402-8.  Back to cited text no. 12
    
13.
Hann SK, Nordlund J, editors. Clinical features of generalized vitiligo. In: Vitiligo: A Monograph on the Basis and Clinical Science. London: Blackwell Science; 2000. p. 35-48.  Back to cited text no. 13
    
14.
Kira M, Kobayashi T, Yoshikawa K. Vitamin D and the skin. J Dermatol 2003;30:429-37.  Back to cited text no. 14
    
15.
Birlea SA, Costin GE, Norris DA. Cellular and molecular mechanisms involved in the action of vitamin D analogs targeting vitiligo depigmentation. Curr Drug Targets 2008;9:345-59.  Back to cited text no. 15
    
16.
Mora JR, Iwata M, von Andrian UH. Vitamin effects on the immune system: Vitamins A and D take centre stage. Nat Rev Immunol 2008;8:685-98.  Back to cited text no. 16
    
17.
Silverberg JI, Silverberg AI, Malka E, Silverberg NB. A pilot study assessing the role of 25 hydroxy vitamin D levels in patients with vitiligo vulgaris. J Am Acad Dermatol 2010;62:937-41.  Back to cited text no. 17
    
18.
Saleh HM, Abdel Fattah NS, Hamza HT. Evaluation of serum 25-hydroxyvitamin D levels in vitiligo patients with and without autoimmune diseases. Photodermatol Photoimmunol Photomed 2013;29:34-40.  Back to cited text no. 18
    
19.
Xu X, Fu WW, Wu WY. Serum 25-hydroxyvitamin D deficiency in Chinese patients with vitiligo: A case-control study. PLoS One 2012;7:e52778.  Back to cited text no. 19
    
20.
Hewison M. An update on vitamin D and human immunity. Clin Endocrinol (Oxf) 2012;76:315-25.  Back to cited text no. 20
    
21.
Marzano AV, Trevisan V, Eller-Vainicher C, Cairoli E, Marchese L, Morelli V, et al. Evidence for vitamin D deficiency and increased prevalence of fractures in autoimmune bullous skin diseases. Br J Dermatol 2012;167:688-91.  Back to cited text no. 21
    
22.
Dahl MV. Imiquimod: A cytokine inducer. J Am Acad Dermatol 2002;47:S205-8.  Back to cited text no. 22
    
23.
Elela MA, Hegazy RA, Fawzy MM, Rashed LA, Rasheed H. Interleukin 17, interleukin 22 and FoxP3 expression in tissue and serum of non-segmental vitiligo: A case- controlled study on eighty-four patients. Eur J Dermatol 2013;23:350-5.  Back to cited text no. 23
    
24.
Aydingöz IE, Bingül I, Dogru-Abbasoglu S, Vural P, Uysal M. Analysis of vitamin D receptor gene polymorphisms in vitiligo. Dermatology 2012;224:361-8.  Back to cited text no. 24
    

What is new?

  • Vitamin D levels are reduced in vitiligo patients
  • Vitamin D level influences the extent of vitiligo
  • Vitamin D receptor expression is reduced in vitiligo lesions compared to normal skin.


    Figures

  [Figure 1], [Figure 2]
 
 
    Tables

  [Table 1], [Table 2], [Table 3]



 

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